The overall goals of this protocol are to collect conditioned medium from mouse embryonic stem cells under serum and feeder free culture conditions and to characterize the biological functions of this medium. This procedure provides insights into the anti senescence activity of soluble factors secreted from mouse embryonic stem cells for the development of safe and cell free therapeutic approach for age related diseases. Demonstrating this procedure will be Dr.Yun-Ui Bae a research professor of my laboratory.
In a biological safety hood, treat a mouse embryonic fibroblast or MEF cell culture with 20 milliliters of MEF medium supplemented with mitomycin C in a 15 centimeter cell culture dish for two hours at 37 degrees celsius and 5%carbon dioxide. At the end of the incubation, wash the cells three times with PBS and detach them with three milliliters of trypsin EDTA for three minutes at 37 degrees celsius. Neutralizing the trypsin with six milliliters of fresh MEF medium when the cells have lifted from the bottom of the plate.
After collecting the cells by centrifugation, re-suspend the pellet in five milliliters of MEF medium and plate two times 10 to the sixth inactivated MEFs per 10 centimeter culture dish in fresh MEF medium. Culture the feeder cells for 24 hours. Then replace the MEF medium with mouse embryonic stem cell medium and seed two times 10 to the sixth mouse embryonic stem cells onto the feeders for at least 48 hours of co culture.
In a biological safety hood, rinse a plate of mouse embryonic stem cells with five milliliters of PBS and detach the cells with trypsin plus EDTA as just demonstrated. After collecting the cells by centrifugation, re-suspend the stem cell pellet in five milliliters of fresh stem cell medium and plate one milliliter of cells per dish in fresh embryonic stem cell medium onto five gelatin coated cell culture dishes. Return the cells to the incubator until the cultures reach 80-85%confluency.
Then rinse the cells in at least eight milliliters of PBS per plate for three 10 minute washes followed by a 24 hour incubation in reduced serum medium. After no more than 24 hours, transfer the supernatant into a 50 milliliter conical tube for centrifugation. Followed by filtration through a 0.2 micrometer bottle top strainer.
To test the effects of the embryonic stem cell conditioned medium by a senescence associated beta galactosidase assay, seed human dermal fibroblasts in a six well plate in a human dermal fibroblast medium. After an overnight incubation at 37 degrees celsius and 5%carbon dioxide, replace half of the medium with fresh mouse embryonic stem cell conditioned medium for another 72 hour culture. On the third day of culture, rinse the cells in two milliliters of PBS per well for two 30 second washes.
Then, fix the cells in 3.7%paraformaldehyde for five minutes at room temperature in a fume hood. After fixing, wash the cells two times with two milliliters of PBS, as just demonstrated and label the cells with one to two milliliters of SA beta gal solution for 17 and half hours at 37 degrees celsius without carbon dioxide. At the end of the staining period, wash the cells two times with PBS as just demonstrated.
Then, counter stain with Eosen for five minutes at room temperature. After another set of PBS washes, image the cells at a 100 times magnification by light microscopy. For cell cycle analysis, seed eight times 10 to the fourth human dermal fibroblast per well in a six centimeter cell culture dish in fresh human dermal fibroblast medium for an overnight incubation at 37 degrees celsius and 5%carbon dioxide.
The next morning, replace half of the medium with mouse embryonic stem cell conditioned medium and return the cells to the incubator. After 24 hours, harvest the cells by trypsin EDTA dissociation followed by two washes in one milliliter of PBS solution. Re-suspend the pellet in 100 microliters of cold PBS solution after the second wash.
Then, fix the cells with the drop wise application at 200 microliters of cold ethanol while vortexing. Store the cells at four degrees celsius for at least an hour. Then, wash them in fresh PBS solution.
After the second centrifugation, treat the pellet with 250 microliters of sodium citrate buffer supplemented with RNase for 30 minutes at 37 degrees celsius without carbon dioxide. At the end of the incubation, label the cells with 250 microliters of sodium citrate buffer containing propidium iodide for 20 minutes. Then, assess the cell cycle stages of each sample by flow cytometry.
For quantitative reverse transcriptase PCR analysis, use an RNA extraction kit to isolate the total RNA from mouse embryonic stem cell conditioned medium treated human dermal fibroblasts according to the manufacturer's protocol. Use a spectrophotometer to quantify the total extracted RNA. Then, formulate reaction mixture containing the oligo deoxythymidine primers and M-MLV reverse transcriptase according to the manufacturer's protocol.
Then add one microgram of the total RNA to the PCR tube containing the reaction mixture. Finally, measure the amplification of the CDNA with a real time PCR machine using the green PCR master mix and the appropriate gene primers. Mouse embryonic stem cells grown under normal mouse embryonic stem cell culture conditions on a MEF feeder cell layer display an oval shape and a shiny appearance.
While mouse embryonic stem cells grown under serum and feeder free culture conditions demonstrate a flattened and irregular morphology. The treatment of the senescent human dermal fibroblast with mouse embryonic stem cell conditioned medium decreases the number of senescent associated betagalactosidase positive cells. Further, cell cycle analysis of these cells reveals that mouse embryonic stem cell conditioned medium treatment dramatically increases the number of cells in the S and G2/M phases while reducing the number of cells in the G0/G1 phase.
In addition, mouse embryonic stem cell conditioned medium treatment decreases the expression of senescence associated genes and of secretary cytokines associated with the senescent phenotype. While attempting this procedure, it is very important to remember to collect the serum and feeder free medium after 24 hours, as a long incubation increases the possibility of cell autolysis or apoptosis by starvation. After watching this video, you should have a good understanding of how to collect serum and feeder free mouse embryonic stem cell conditioned medium for a cell free approach.
Don't forget that working with reagent such as paraformaldehyde can be extremely hazardous, and that precaution, such as handling this reagent in a fume hood, should always be taken while performing this procedure.
